J O U R N A L O F T H E M E C H A N I C A L B E H AV I O R O F B I O M E D I C A L M A T E R I A L S 5 ( 2 0 1 2 ) 1 – 8

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journal homepage: www.elsevier.com/locate/jmbbm

Research paper

Overexpression of DMP1 accelerates mineralization and alterscortical bone biomechanical properties in vivo✩

Ankush Bhatiaa, Michael Albazzazb, Alejandro A. Espinoza Oríasc, Nozomu Inouec,Lisa M. Millerd,e, Alvin Acerboe, Anne Georgeb, Dale R. Sumnera,c,∗

aDepartment of Anatomy and Cell Biology, Rush Medical College, 600 S Paulina St., Rm 507, Chicago, IL, 60612, USAbDepartment of Oral Biology, University of Illinois at Chicago, 801 S Paulina St., Chicago, IL, 60612, USAcDepartment of Orthopedic Surgery, Rush University Medical Center, 1611 W. Harrison St., Chicago, IL, 60612, USAdNational Synchrotron Light Source, Brookhaven National Laboratory, 75 Brookhaven Avenue, Bldg 725B, Upton, NY, 11973, USAeDepartment of Biomedical Engineering, State University of New York at Stony Brook, HSC T18, Rm 030, Stony Brook, NY, 11794, USA

A R T I C L E I N F O

Article history:

Received 13 March 2011

Received in revised form

19 August 2011

Accepted 23 August 2011

Published online 29 September 2011

Keywords:

DMP1

Biomineralization

MicroCT

Mechanical testing

Transgenic

Cortical bone

A B S T R A C T

Dentin matrix protein-1 (DMP1) is a key regulator of biomineralization. Here, we examine

changes in structural, geometric, and material properties of cortical bone in a transgenic

mouse model overexpressing DMP1. Micro-computed tomography and three-point bending

were performed on 90 femora of wild type and transgenic mice at 1, 2, 4, and 6 months.

Fourier transform infrared imaging was performed at 2 months. We found that the

transgenic femurs were longer (p < 0.01), more robust in cross-section (p < 0.05), stronger

(p < 0.05), but had less post-yield strain and displacement (p < 0.01), and higher tissue

mineral density (p < 0.01) than the wild type femurs at 1 and 2 months. At 2 months, the

transgenic femurs also had a higher mineral-to-matrix ratio (p < 0.05) and lower carbonate

substitution (p < 0.05) compared to wild type femurs. These findings indicate that increased

mineralization caused by overexpressing DMP1 led to increased structural cortical bone

properties associated with decreased ductility during the early post-natal period.c⃝ 2011 Elsevier Ltd. All rights reserved.

e

,

a

d

1. Introduction

Biomineralization of bone and dentin is a highly regu-lated process that is still not completely understood. Dur-ing this process, it is known that both osteoblasts and

✩ Funding sources: the NIH Grants R01DE11657, T32AR052272, andesign; in the collection, analysis, and interpretation of data; in thpublication.

∗ Corresponding author at: Department of Anatomy and Cell BiologyUSA. Tel.: +1 312 942 5511; fax: +1 312 942 5744.

E-mail addresses: ankush_bhatia@rush.edu, ankushbhatia1@gmalejandro_espinoza@rush.edu (A.A. Espinoza Orías), nozomu_inoue@(A. Acerbo), anneg@uic.edu (A. George), rick_sumner@rush.edu (D.R.

1751-6161/$ - see front matter c⃝ 2011 Elsevier Ltd. All rights reservedoi:10.1016/j.jmbbm.2011.08.026

d the Grainger Foundation. Funding sources had no role in studywriting of the report; and in the decision to submit the paper for

Rush Medical College, 600 S Paulina St., Rm 507, Chicago, IL, 60612,

il.com (A. Bhatia), anneg@uic.edu (M. Albazzaz),rush.edu (N. Inoue), lmiller@bnl.gov (L.M. Miller), acerbo@bnl.govSumner).

odontoblasts secrete type I collagen and non-collagenousproteins (NCPs). In bone, the NCPs help mineralize theosteoid and can be divided into four general groups:proteoglycans, glycosylated proteins, glycosylated proteinswith potential cell-attachment activities, and γ-carboxylated

.

2 J O U R N A L O F T H E M E C H A N I C A L B E H AV I O R O F B I O M E D I C A L M A T E R I A L S 5 ( 2 0 1 2 ) 1 – 8

Table 1 – Sample size distribution.

Months Wild type Transgenic Totals

1 13 17 302 9 15 244 8 8 166 10 10 20

Totals 40 50 90

proteins (Robey and Boskey, 2006). Each group containsa number of proteins, all of which are being studied tounderstand their roles in mineralization. Dentin matrixprotein-1 (DMP1) is a member of the small integrin-bindingligand, n-glycosylated protein and other glycoproteins withcell attachment activity (SIBLING) family, along with osteo-pontin (OPN), bone sialoprotein (BSP), matrix extracellularphosphoglycoprotein (MEPE), and dentin sialophosphopro-tein (DSPP) (Fisher and Fedarko, 2003; Fisher et al., 2001).DMP1 was first discovered in dentin (George et al., 1993), butit was later found in bone and other tissues such as the kid-ney, pancreas, brain, and salivary and eccrine sweat glands(D’Souza et al., 1997; Hirst et al., 1997; MacDougall et al., 1998;Ogbureke and Fisher, 2004, 2005, 2007; Terasawa et al., 2004).While this protein has been found in various tissues, DMP1’smost influential function is its role as a key regulator in thecomplex process of biomineralization.

In vitro, DMP1 was first shown to be involved in mineral-ization when cells overexpressing DMP1 showed accelerateddifferentiation and mineralization (Narayanan et al., 2001).Later, DMP1 was shown to nucleate hydroxyapatite (He et al.,2003). In vivo, DMP1 knockout mice have delayed secondaryossification centers, enlarged growth plates with dramatic ex-pansion of hypertrophic chondrocyte zones, and short limbs(Feng et al., 2006; Ling et al., 2005; Ye et al., 2005). Additionally,deficiencies in mineralization in these mice contribute to os-teomalacia (Feng et al., 2006; Ling et al., 2005; Ye et al., 2005).

Overexpressing DMP1 in vivo through a gain-of-functionapproach confirmed the earlier in vitro studies and showedaccelerated mineralization, chondrocyte hypertrophy inosseous tissues, and a five-fold increase of DMP1 expression(RNA and protein) in both teeth and bone when comparedto liver in the transgenic mice (Albazzaz et al., 2009). In thisstudy, we use this transgenic model to test if overexpressingDMP1 leads to altered structural, geometric or materialproperties during post-natal maturation of the appendicularskeleton. Micro-computed tomography (µCT), three-pointbending tests, and Fourier transform infrared imaging (FTIRI)demonstrated accelerated mineralization and less ductilemechanical behavior during rapid phases of longitudinalgrowth in the mouse femur.

2. Materials and methods

2.1. Animals

This study used 90 mice that were divided among wild typeand transgenic animals at 1, 2, 4, and 6 months (Table 1).CD-1 mice from Charles River Laboratories overexpressedDMP1 through preparation of a cytomegalovirus (CMV)

— DMP1 construct that induces ubiquitous expressionof DMP1 (Albazzaz et al., 2009). The mice were housedin a controlled temperature environment with daylightphotoperiod-controlled lighting. They were allowed freeaccess to food and water. The experiments were conductedaccording to the University of Illinois at Chicago animalcare-approved protocols, and animals were maintainedin accordance with the Institute of Laboratory AnimalResearch’s Guide for the Care and Use of Laboratory Animals.

Following necropsy at respective ages, right femurs weredissected, freed of adherent soft tissue, and refrigeratedin saline at 4 ◦C for subsequent analysis including µCT,biomechanical testing, and FTIRI. The femurs were measuredalong the proximal–distal direction using a digital caliper(Control Company, Texas). Genders were combined andrepresented consistently at each group and age. Of 94 femursinitially included, four were eliminated due to errors inmechanical testing, resulting in a sample size of 90 for allanalyses. Representative contralateral femurs at 2 monthswere analyzed by FTIRI (n = 4 per group).

2.2. µCT

Desktop micro-computed tomography (µCT 40, Scanco Medi-cal AG, Basserdorf, Switzerland) was used to nondestructivelyevaluate bone cross-sectional geometric properties. Femurswere scanned submerged in saline solution and oriented or-thogonally to the axis of the X-ray beam, using 55 kVp, 145 µA,and an integration time of 300 ms to collect data from a1.4 mm-long segment of the mid-diaphysis. Scans were per-formed at a nominal resolution of 12 µm. Sigma gauss was 0.8and support gauss was 1. All samples were thresholded at 260and the manufacturer’s software was used to determine aver-age cortical thickness (Ct.Th; mm), cortical bone area (Ct.Ar;mm2), total cross-sectional area inside the periosteal enve-lope (Tt.Ar; mm2), medullary area (Ma.Ar; mm2), the momentof inertia about the medial–lateral axis (Iml;mm4), maximumand minimum moments of inertia (Imax;mm4, Imin;mm4),section modulus (Imax/Cmax, Imin/Cmin), and polar momentof inertia (J; mm4). In addition, the tissue mineral density ofthe cortical bone was determined using the manufacturer’scalibration phantom and software set at 1200 mgHA/ccm.We use the standard nomenclature and reporting guidelinesfor assessment of bone microstructure in rodents with µCT(Bouxsein et al., 2010).

2.3. Biomechanical testing

Three-point bending was performed in position controlusing a custom-made materials testing machine, whichconsists of a linear actuator with optical encoder (Digitseries, UltraMotion), a 250 N load cell (Entran, EL series,Measurement Specialties, Hampton, VA), a custom-madespecimen holder, and a water chamber. The test wasperformed in 9 ml of physiological saline. Since femur lengthchanged with each age, the span lengths (L) were varied perbone and were normalized to approximately 67% of the totalbone length. Therefore, a set of 8 custom-made specimenholders were constructed with span lengths of 7, 8, 9, 10,10.5, 11, 11.5, and 12 mm. The cross-head speed of 0.2 mm/s

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was held constant throughout and force–displacement datawere recorded at a sampling rate of 100 Hz. Load was appliedin the anterior–posterior direction, midway between the twosupport points in the exact region that underwent µCT. Fromthe force–displacement curve, bending stiffness (S; N/mm),yield moment (FY ; N), maximum moment (FU; N), energy tofailure (mJ), energy to yield (mJ), post-yield energy to failure,ultimate displacement (D), pre-yield displacement, and post-yield displacement (DPY) were determined. Bending stiffnesswas defined as the slope of the initial linear portion of theload displacement curve. Maximum force was defined as thepeak of the curve on the y-axis. Yield moment was definedusing a 0.015 mm offset parallel to the stiffness (Schrieferet al., 2005). Energy to yield was defined as the area underthe curve until the yield point. Energy to failure was definedas the area under the entire curve. Post-yield energy to failurewas defined as the area under the curve between the yieldand failure points. Ultimate displacement was defined asthe total distance traveled by the load starting from 0.4 Nuntil failure. Pre-yield and post-yield displacements were alsocalculated.

Since the bones varied in size, the actual span betweenloading points were of various sizes. Fortunately, this canbe normalized in terms of bending moment. For three-pointbending, themaximum bendingmoment is registered at mid-span on the test specimen, allowing normalization of datain terms of bending strain, which is a more appropriate wayof describing data regardless of sizes. A similar analysis wasperformed by Draper and Goodship for four-point bendingtests (Draper and Goodship, 2003). Derivation of equationsfrom first principles (Euler–Bernoulli beam theory) is includedin the Appendix.

From these structural properties and the geometricproperties from µCT, material properties were calculated asfollows:

σU = FU

Lc

4IML

εPr e−yield =

6DPr e−yieldd

L2

εPost−yield =

6Dpost−yieldd

L2

E = S

L3

48IML

u = U

3D2

4IMLL

,

where σU is the ultimate stress (MPa), ε is the strain, E isYoung’s modulus (GPa), u is themodulus of toughness (MPa), Lis the length between the two spans, c is one-half of the bonediameter measured perpendicular to the neutral axis (mm), dis the bone diameter at the mid-shaft (mm), U is the energyto failure, and the other terms are as defined (Schriefer et al.,2005) above.

2.4. Fourier transform infrared imaging

Femurs were embedded in methyl methacrylate, 2 mm thicktransverse sections were cut at the mid-diaphysis with adiamond-tipped blade (Isomet 5000, Buehler, Lake Bluff, IL),

and then ground and polished with a final finish using 9 µmred diamond suspension paste (Phoenix 4000, Buehler, LakeBluff, IL) to prepare a flat smooth surface. Fourier TransformInfrared Imaging (FTIRI) was performed using a Bruker Vertex80V Step-Scan FTIR spectrometer and Hyperion 3000 infraredmicroscope equipped with a 64 × 64 pixel focal plane arraydetector. Images were collected in reflection mode from thesurface of the polished bone with 4 cm−1 resolution, 256scans/image and an effective pixel resolution of 20 × 20 µm2.Spectra were converted to absorbance using a custom,Matlab-based Kramers–Kronig transformation routine.

Spectra were analyzed for three characteristic peakswhich have been associated with specific chemical com-ponents of bone tissue. Protein (1600–1700 cm−1, base-line: 1300–1800 cm−1), carbonate (1414–1424 cm−1, baseline:1300–1800 cm−1) and phosphate (900–1200 cm−1, baseline:900–1200 cm−1) contents were determined by area inte-gration (Miller et al., 2007). Mineral-to-matrix ratio wascalculated as the phosphate/protein ratio and carbonate sub-stitution was calculated as the carbonate/phosphate ratio.The ratio of nonreducible/reducible collagen cross-linkingwas determined from a peak height ratio of 1660/1690 cm−1

(baseline: 1300–1800 cm−1) (Paschalis et al., 2001) andcrystallinity was determined from a peak height ratio of1020/1030 cm−1 (baseline: 900–1200 cm−1) (Paschalis et al.,1996).

2.5. Statistical analysis

Gender differences were examined and found to be minimal,so this factor was ignored in the statistical analyses. ForµCT and biomechanical testing, two-way analyses of variance(ANOVA) were used to evaluate differences between thefemurs from the four ages (1, 2, 4 and 6 months) and thetwo groups (wild type and transgenic). Where group wassignificant or there was a significant group-by-age interactionterm, independent Student t-tests were used to assessdifferences between transgenic and wild type animals ateach age. For the FTIRI analysis at 2 months, we used theMann–Whitney U test to assess statistical difference due tothe smaller sample size. Significance was noted at p < 0.05.All statistical analyses were conducted using SPSS (SPSS 15.0for Windows; SPSS, Chicago, IL) statistical software. All dataare represented as mean ± standard deviation.

3. Results

3.1. Geometric properties (Fig. 1)

Transgenic mouse femurs were significantly longer comparedto wild-type mouse femurs at 1 and 2 months (Table 2).However, by 4 and 6 months, there were no differences inbone length. The transgenic mouse femurs had significantlygreater cross-sectional geometric properties than the wild-type mouse femurs at one month, including Ct.Ar, Tt.Ar,Ma.Ar, Imax, Imin, Imax/Cmax, Imin/Cmin, and J (Table 2). At2 months, Tt.Ar, Imin, Imin/Cmin, and J were greater in thetransgenic femurs. At 4 months, Imax was the only geometricproperty that was significantly greater in the transgenic

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Table 2 – Geometric (length and mid-shaft femoral cross-sectional) properties (mean ± standard deviations).

Age (months)1 2 4 6 ANOVA

Femur length TR 12.56* (.69) 15.44** (.67) 16.22 (.43) 16.80 (.46) a,b,c(mm) WT 11.62 (1.11) 14.01 (1.32) 15.92 (.71) 17.06 (.46) a,b,cCt.Ar (mm2) TR 0.64* (.06) 0.99 (.10) 1.02 (.13) 1.14 (.14) a,b

WT 0.55 (.12) 0.91 (.09) 0.92 (.12) 1.06 (.10)Ct.Th (mm) TR 0.18 (.01) 0.24 (.02) 0.26 (.02) 0.26 (.01) a

WT 0.16 (.03) 0.23 (.02) 0.24 (.02) 0.26 (.02)Tt.Ar (mm2) TR 1.32** (.07) 1.81* (.16) 1.83 (.21) 2.10 (.40) a,b

WT 1.19 (.17) 1.65 (.14) 1.69 (.22) 1.90 (.25)Ma.Ar (mm2) TR .70** (.04) .82 (.10) .78 (.13) .96 (.27) a,b

WT .65 (.07) .74 (.06) .70 (.10) .84 (.21)Imax(mm4) TR 0.13* (.02) 0.27 (.06) 0.28* (.10) 0.35 (.13) a,b

WT 0.10 (.04) 0.23 (.04) 0.21 (.05) 0.28 (.07)Imin(mm4) TR 0.08** (.01) 0.16** (.02) 0.15 (.03) 0.23 (.08) a,b

WT 0.06 (.02) 0.13 (.02) 0.14 (.03) 0.19 (.04)Imax/Cmax(mm3) TR 0.18* (.02) 0.29 (.05) 0.30 (.07) 0.36 (.09) a,b

WT 0.14 (.04) 0.26 (.04) 0.25 (.05) 0.32 (.05)Imin/Cmin(mm3) TR 0.14** (.01) 0.24** (.03) 0.23 (.03) 0.30 (.06) a,b

WT 0.11 (.03) 0.20 (.03) 0.21 (.03) 0.27 (.04)J(mm4) TR 0.21* (.03) 0.43* (.08) 0.43 (.13) 0.59 (.21) a,b

WT 0.17 (.05) 0.36 (.07) 0.34 (.08) 0.48 (.11)

* p < 0.05, ** p < 0.01, *** p < 0.001, vs. WT at same age. a, Two-way ANOVA significant for age (p < 0.05); b, Two-way ANOVA significant forgroup (p < 0.05); c, Two-way ANOVA significant for the age-by-group interaction (p < 0.05). TR = Transgenic, WT = Wild type. Ct.Ar = corticalbone area, Ct.Th = average cortical thickness, Tt.Ar = total cross-sectional area inside the periosteal envelope, Ma.Ar = medullary area, Imax =

maximum moment of inertia, Imin = minimum moment of inertia, Imax/Cmax, Imin/Cmin = section modulus, J = polar moment of inertia.

Fig. 1 – Geometrical properties: Femur length and polarmoment of inertia (mean ± standard deviations). TR =

transgenic mice, WT = wild type mice, ∗p < 0.05, ∗∗p < 0.01for TR vs. WT mice at a given age.

femurs compared to the wild type femurs. There were nodifferences in any of these geometric properties betweentransgenic and wild type femurs at 6 months. There were nodifferences in cortical thickness at any time point.

3.2. Structural mechanical properties (Fig. 2)

At 1 month, transgenic mouse femurs had higher yieldand maximum moments compared to the wild type mousefemurs (Table 3). At 1 and 2 months, it took significantlyless energy to fracture the transgenic femurs and it took lessenergy to break the transgenic femurs in the post-yield phase,although the energy to yield was significantly higher in thetransgenic femurs at 2 months. In addition, the transgenicfemurs had less ultimate displacement and less post-yielddisplacement at 1 and 2 months than the wild type femurs.There were no differences in bending stiffness or pre-yield

Fig. 2 – Whole bone mechanics: Maximum force andenergy to failure (mean ± standard deviations). TR =

transgenic mice, WT = wild type mice, ∗p < 0.05, ∗∗p < 0.01for TR vs. WT mice at a given age.

displacement at any time point and there were no differencesin any structural mechanical properties between transgenicand wild type femurs at either 4 or 6 months.

3.3. Material properties (Fig. 3)

Transgenic femurs had significantly higher tissue mineraldensity and lower post-yield strain andmodulus of toughnessat 1 and 2 months than the wild type femurs (Table 4).Differences in Young’s modulus, ultimate stress, and pre-yield strain were not significant between the transgenic andwild type femurs at 1 and 2 months. At 4 and 6 months,there were no significant differences in anymaterial property.At 2 months, transgenic femurs had significantly highermineral-to-matrix ratios and lower carbonate substitution ascompared to wild type femurs (Fig. 4, Table 5). There were nodifferences in mineral crystallinity or collagen cross-linkingas assessed by FTIRI.

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Table 3 – Structural mechanical properties (mean ± standard deviations).

Age (months)1 2 4 6 ANOVA

Bending Stiffness TR 40.30 (6.68) 56.49 (11.01) 64.04 (15.12) 75.37 (19.01) a(N/mm) WT 28.79 (13.79) 56.94 (12.85) 61.74 (10.90) 68.16 (18.51)Maximum moment (N) TR 15.91* (2.84) 40.15 (5.50) 44.19 (6.57) 55.87 (8.08) a,b

WT 12.6 (6.07) 34.36 (11.53) 41.64 (6.22) 54.04 (8.78)Yield moment (N) TR 12.06* (3.01) 31.07* (3.92) 36.04 (5.89) 50.42 (6.86) a,b

WT 9.08 (4.94) 24.33 (9.86) 34.27 (4.62) 43.26 (10.99)Total Energy (mJ) TR 3.26* (1.00) 6.83* (2.51) 5.29 (2.40) 5.85 (2.63) a,b

WT 5.69 (3.24) 9.39 (1.88) 4.69 (1.25) 6.74 (1.80)Energy to Yield (mJ) TR 0.49 (.14) 1.52* (.55) 1.56 (.64) 2.47 (.64) a,b

WT 0.39 (.27) 0.97 (.39) 1.63 (.53) 1.88 (.56)Post-yield Energy (mJ) TR 2.77* (.95) 5.31** (2.15) 3.73 (2.15) 3.38 (2.83) a,b,c

WT 5.29 (3.13) 8.42 (1.91) 3.05 (1.56) 4.86 (1.69)Ultimate Displacement TR 0.55*** (.16) 0.64*** (.21) 0.48 (.15) 0.47 (.12) a,b,c(mm) WT 1.34 (.52) 0.94 (.20) 0.46 (.11) 0.55 (.16)Pre-yield TR 0.14 (.02) 0.26 (.09) 0.22 (.05) 0.28 (.06) a,b,cDisplacement (mm) WT 0.14 (.05) 0.18 (.03) 0.23 (.06) 0.24 (.05)Post-yield TR 0.41*** (.16) 0.38*** (.15) 0.26 (.13) 0.19 (.15) a,b,cDisplacement (mm) WT 1.20 (.53) 0.76 (.19) 0.23 (.14) 0.31 (.14)

* p < 0.05, ** p < 0.01, *** p < 0.001, vs. WT at same age. a, Two-way ANOVA significant for age (p < 0.05); b, Two-way ANOVA significant for group(p < 0.05); c, Two-way ANOVA significant for the age-by-group interaction (p < 0.05). TR = Transgenic, WT = Wild type.

Table 4 – Material Properties (mean ± standard deviations).

Age (months)1 2 4 6 ANOVA

Tissue MineralDensity

TR 1045.32** (13.99) 1194.43*** (17.99) 1270.07 (38.56) 1302.30 (25.90) a,b,c

(mgHA/cm3) WT 1014.25 (33.18) 1149.95 (13.87) 1262.49 (13.84) 1295.24 (20.11)Ultimate Stress TR 104.23 (13.67) 157.02 (14.41) 187.79 (28.17) 185.80 (36.11) a(MPa) WT 98.83 (30.62) 148.97 (22.06) 182.59 (22.17) 177.57 (22.78)Pre-yield Strain TR 0.030 (0.005) 0.040 (0.012) 0.030 (0.007) 0.042 (0.008) a

WT 0.031 (0.005) 0.036 (0.009) 0.032 (0.006) 0.035 (0.010)Post-yield Strain TR 0.05** (.04) 0.03** (.01) 0.02 (.01) 0.02 (.01) a,b,c

WT 0.14 (.09) 0.07 (.03) 0.02 (.01) 0.02 (.01)Young’s Modulus TR 5.20 (1.55) 7.62 (1.98) 11.20 (2.41) 9.49 (2.49) a(GPa) WT 4.37 (2.00) 6.47 (1.98) 10.37 (2.11) 9.63 (3.37)Modulus of TR 2.25* (4.87) 1.31** (1.74) 0.57 (.59) 0.34 (.22) a,b,ctoughness (MPa) WT 16.68 (19.18) 4.15 (1.76) 0.49 (.41) 0.61 (.49)

* p < 0.05, ** p < 0.01, *** p < 0.001,vs. WT at same age. a, Two-way ANOVA significant for age (p < 0.05); b, Two-way ANOVA significant for group(p < 0.05); c, Two-way ANOVA significant for the age-by-group interaction (p < 0.05). TR = Transgenic, WT = Wild type.

4. Discussion

Our objective was to determine if the overexpression of DMP1affected bone geometry, structural (whole bone) mechanicalor material properties. Overall, we found effects at 1 and2 months that were no longer present at 4 and 6 months.Specifically, overexpression of DMP1 was associated withmore rapid longitudinal and cross-sectional bone growth,increased strength combined with decreased post-yieldenergy dissipation as a structure, and depressed toughnessand increased density as a material. Thus, even though thetransgenic mouse femurs were stronger, they also requiredless energy to fracture. The transgenic femurs demonstratedless post-yield ability to withstand deformation or strainduring mechanical testing; implying that overexpression ofDMP1 caused the cortical bone to behave in a less ductilemanner.

Fig. 3 – Material properties: Material density andpost-yield strain (mean ± standard deviations). TR =

transgenic mice, WT = wild type mice,∗∗p < 0.01, ∗∗∗p < 0.001 for TR vs. WT mice at a given age.

Several factors may contribute to this bone pheno-

type. Overexpressing DMP1 has been shown to accelerate

6 J O U R N A L O F T H E M E C H A N I C A L B E H AV I O R O F B I O M E D I C A L M A T E R I A L S 5 ( 2 0 1 2 ) 1 – 8

Fig. 4 – µCT and FTIRI images. Examples of µCT cross-sections of wild type (WT) and transgenic (TR) femurs withrespective FTIRI images, including mineralization, carbonate substitution, mineral crystallinity, and collagen cross-linking.

mineralization in vitro (Narayanan et al., 2001). The presentstudy showed that the cortical bone had a significantly highertissue mineral density and mineral-to-matrix ratio and thatthese changes in material properties were associated withless ductile mechanical behavior at a material and, to someextent, structural level. This association between increasedmineralization and less ductile bone behavior is consistentwith many previous studies of the role of mineralization indetermining how bone behaves mechanically (Currey, 1962).While we have focused largely on the inorganic phase (tis-sue mineral density), other aspects of mineral quality or dif-ferences in the organic phase may also contribute to DMP1’seffects on the bone’s mechanical behavior (Ling et al., 2005).

The bone phenotype observed in the present study,accelerated mineralization and less ductile mechanicalbehavior in mice overexpressing DMP1, is consistent withearlier studies in the same mouse model in whichoverexpression of DMP1 was associated with acceleratedmineralization, as assessed by whole mount staining, up toone month post natally (Albazzaz et al., 2009). Interestingly,opposite effects were observed by previous investigators inmice with DMP1 expression knocked out (He et al., 2003; Hirstet al., 1997; Ling et al., 2005). Together, these studies suggestthat lack of DMP1 inhibits mineralization and depresses bonegrowth while increased DMP1 accelerates mineralizationand stimulates bone growth. While the skeleton does notcompensate for the absence of DMP1 post-natally (Hirstet al., 1997), the phenotypic effects of DMP1 overexpressionobserved in the present study were evident only during rapidbone growth but did not persist as the animals approachedskeletal maturity. While femur length was increased early on,it is unlikely that the stimulation in bone growth at 1 and 2months is related to the size of themice because earlier worksin this model showed no statistical difference of weight vs ageor group (unpublished).

As the role of DMP1 in mineralization is furtherunderstood, various mechanisms may explain these changesin bone phenotype that occur only during rapid phases ofgrowth. One possible explanation revolves around the role

Table 5 – FTIR (mean ± standard deviations).

2 monthsTR WT

Mineral:Matrix 2.40* (0.17) 1.82 (0.05)Crystallinity 1.94 (0.24) 2.01 (0.29)CollagenCross-Linking

2.01 (0.11) 2.17 (0.23)

CarbonateSubstitution

0.023* (0.002) 0.031 (0.003)

*p < 0.05, ** p < 0.01, *** p < 0.001, vs. WT at 2 months. TR =

Transgenic, WT = Wild type.

of DMP1 expression in preosteoblasts, late osteoblasts andosteocytes (Eapen et al., 2010; Narayanan et al., 2003). Inthe preosteoblasts, one of the co-author’s laboratory hasdemonstrated that DMP1 is in the nucleus and acts as atranscription factor (Narayanan et al., 2003). Further, theGeorge laboratory has recently demonstrated that DMP1 cantrigger Runx 2 expression (Eapen et al., 2010). At later stagesduring osteoblast differentiation, DMP1 is exported out to thematrix and initiates hydroxyapatite nucleation (Narayananet al., 2003). Based on these observations in vitro, we suggestthat during early stages of development, DMP1 can trigger theosteoblast differentiation cascade. At later stages there mightbe other factors that inhibit this process.

A limitation in this study is the ability to estimate tissuelevel mechanical properties using beam theory. The value ofYoung’s modulus was relatively low (4–11 GPa) in our study,and upon reviewing the literature, we have found a highvariation of reported values of E. The reported values for E aredependent on the method employed. Raum et al., reportedvalues in the range of mid teens (16.3 + / − 1.9 GPa) whenusing ultrasound and acoustic microscopy (Raum et al., 2007).Somerville et al. reported even higher numbers at 22.3 GPa formales (Somerville et al., 2004). Van Lenthe et al. used a microfinite element model and arrived at values of approximately12.0 GPa for B6 WT mice (van Lenthe et al., 2008) andemphasized the underestimation using beam theory as a

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motivation for using micro finite element analysis. Chattahet al. explored the influence of freezer storage time andobtained values between 8.6 and 10.9 GPa for BL6mice femursusing Electronic Speckle Interferometry (Chattah et al., 2009).Brodt et al. reported E values in the neighborhood of 10 GPafor 12 and 16 week old mice (Brodt et al., 1999). One ofthe co-authors found an even lower E value for Balb/C WTmice femurs: 6.57 GPa (Espinoza Orias et al., 2008). Thereseems to be sensitivity to the testing method, but our resultsstill are in general concordance with values found in theliterature. The experiments were carefully carried out and ouranalysis method is outlined in the Appendix. Clearly, newertechnology is finding some variation, enabling researchers tofine tune the analysis methods. This would not necessarilymean that the results using beam theory, especially forcomparative purposes, are wrong; however if exact values areneeded, then micro modeling with the finite element methodmay be preferable.

Various pharmacological and mechanical perturbationsare known to alter the response of bone cells at theendocortical and periosteal surfaces differently. With DMP1overexpression, both the endocortical and periosteal areasincreased at 1 and 2 months, implying increased net boneresorption at the endocortical surface and increased netbone formation at the periosteal surface. Further histologicalevaluation is needed to understand the relative importanceof bone formation and resorption to these changes at theendocortical and periosteal surfaces.

Since the main goal of this study was to assess theeffect of DMP1 overexpression on cortical bone, we combinedmales and females to increase our sample size. However, wedid conduct initial three-way analyses of variance with age,group, and gender, and did not find any gender effects. Sincemales and females were equally divided at each age andgroup, it is unlikely that gender played a significant role inthis study. Therefore, we decided to ignore gender and focuson age and genotype effects. Nevertheless, without having amuch larger sample, it is not possible to conclude that genderhas no influence on the skeletal response to overexpressionof DMP1.

We found a number of age-by-group interactions. Theseusually occurred when there were significant differencesbetween wild type and transgenic animals at 1 and 2 months,but not at 4 and 6 months. Although the differences betweenwild type and transgenic animals at 4 and 6 months weresmaller then at 1 and 2 months, it is not possible to concludethat they were not actually present as the study was notadequately powered to detect such small differences. In anycase, however, it is safe to conclude that the effect of DMP1overexpression was at least much diminished at 4 and 6months and for some variables was completely eliminated.Thus, the value of examining different ages when trying toassess a skeletal phenotype in transgenic mouse models isclear.

The data of the present study indicate that DMP1overexpression engendered differences in bone geometry,structural mechanical properties and material propertiesduring phases of rapid growth and that these effectsdiminished as the animals approached skeletal maturity.Thus, DMP1 overexpression is associated with more highly

mineralized cortical bone which behaves in a less ductilemanner and with increases in cross-sectional properties. Thenet effect of these geometric and material effects is increasedstrength coupledwith depressed post-yield energy absorptionat the structural (whole bone) level during phases of rapidlongitudinal bone growth.

Appendix. Supplementary data

Supplementary material related to this article can be foundonline at doi:10.1016/j.jmbbm.2011.08.026.

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